Transcript Deth PrEP and PEP

“Prenatal and Postnatal Epigenetic Programming
(PreP and PEP):
Implications for GI, Immune and Neuronal
Function in Autism”
Richard Deth, PhD
Northeastern University
Boston, MA
Overview
- Oxidation and antioxidant metabolism
- Autism: An epigenetic disorder
Prenatal epigenetic programming (PREP)
Postnatal epigenetic programming (PEP)
-Brain-specific redox features
- Redox signaling in the immune response
- Gluten and casein-derived opiate peptides
Viewing Life Through Redox Glasses
Ox
ida
tio
n
Re
du
cti
on
Oxidation: Loss of an electron
Reduction: Gain of an electron
Life:
The evolved ability to resist oxidation
Death:
The inevitable outcome of oxidation
Evolution:
Gradual acquisition and manifestation of novel adaptive strategies
to survive oxidation
Development:
Programmed and progressive changes in gene expression,
driven by changes in oxidation status, via epigenetic mechanisms
As oxygen became more plentiful,
evolution was driven by the ability
to adapt and to resist oxidation.
Earliest life appears arose at
hydrothermal vents emitting
hydrogen sulfide.
H2S
From Paul G. Falkowski; Science 311 1724 (2006)
EVOLUTION = LAYER UPON LAYER OF USEFUL
ADAPTIVE RESPONSES TO OFFSET
THE THREAT OF OXIDATION
The ability to control
oxidation is at the
core of evolution
Each addition is
strengthened because
it builds on the
solid core already
in place.
Ergo:
1. Life, from beginning to end, depends upon sufficient levels of
antioxidant.
2. Disorders and diseases commonly reflect an interference
with the capacity to resist oxidation.
3. Agents or organisms which interfere with antioxidant capacity
will disrupt normal development and cause diseases.
4. Metabolic treatments that restore and sustain antioxidant
capacity will be effective in reversing many disorders and diseases.
The available level of antioxidant (GSH)
controls the level of aerobic metabolism
and ATP formation = Redox Signaling
Glucose
NADPH
Selenoproteins
Antioxidant
Supply
Glutathione
NADH+
O2 + 4H+
2H2O + ATP
formation
Reactive
Oxygen ROS
Species
[Antioxidant]
Glutathionylation
of Complex I
Oxidative
Cellular
Damage
No Damage
Water
Oxygen
e.g ROS
e-
Reducing equivalents
are passed from
selenium to sulfur and
from sulfur to oxygen
Sulfur
e.g. GSH
e-
NADPH
Selenium
e.g. Thioredoxin
Reductase
Illustrations from: Bentor, Yinon. Chemical Element.com
Hydrogen
(Reducing Equivalent)
Autism is associated with inadequate levels
of glutathione and impaired methylation
28%↓
36%↓
38%↓
Eleven studies showing significantly lower GSH in autism
Authors
(Reference)
Control n
Autistic
n
GSH
status
Additional related findings
33
20
46% ↓
↓GSH/GSSG, ↓SAM/SAH, ↓cysteine
73
80
32% ↓
↓GSH/GSSG, ↓SAM/SAH, ↓cysteine
10
36%↓
↓cysteine
55
43
21% ↓
↓SAM, ↓cysteine
Pasca et al.
(2009)
13
15
33% ↓
↓cysteine
Pastural et al.
(2009)
Al-Gadani et
al.
(2009)
Melnyk et al.
(2011)
James et al.
(2009)
Geier et al.
(2009)
12
15
35% ↓
↓cysteine
30
30
27%↓
↑lipid peroxides, ↓vitamin E, ↓SOD, ↓GPx
40
40
29%↓
42
40
28%↓
↓GSH/GSSG, ↓SAM/SAH, ↓cysteine,
↓DNA methylation
↓GSH/GSSG, ↓SAM/SAH, ↓cysteine
120
28
24% ↓
↓cysteine
Lab-based
normal
values
28
24% ↓
↑GSSG, ↓cysteine, ↓taurine, ↓sulfate
James et al.
(2004)
James et al.
(2006)
Geier and
Geier
(2006)
Adams et al.
(2011)
Geier et al.
(2009)
Lab-based
normal
values
The available level of intracellular cysteine
is rate-limiting for GSH synthesis
GLUTATHIONE (GSH)
Glycine
γ- Glutamylcysteine
Glutamate
[Cysteine]
Cysteine for glutathione synthesis can be provided by either
transsulfuration of homocysteine or by uptake from outside the cell
Cysteine
NEURONAL
CELL
Cysteinylglycine
GSSG
Glial Cells
(Astrocytes)
GSH
GSH
γ-Glutamylcysteine
Glutathione
Synthesis
Cysteine
Transsulfuration
Cystathionine
Adenosine
SAH
HCY
MethylTHF
Methionine
Synthase
>150
Methylation
Reactons
THF
SAM
MET
ATP
PP+Pi
Dietary protein
(-)
Absorption and distribution of cysteine/cystine
GI TRACT
BLOOD
BRAIN
Blood
Brain
Barrier
GI
Epithelial
Cells
EAAT3
Cysteine
GSH
Cysteine
Astrocytes
Cystine
GSH
GSH
Cysteine
Cystine
Methionine
Cysteine
Cystine EAAT3
GSH
Dietary
Protein
Cysteine
Neurons
LIVER
Cysteine
Cystine
The GI tract is a critical site for
immune cell activation, especially
during postnatal development.
Decreased cysteine uptake can
promote oxidative stress and
inflammation, resulting in
epigenetic effects which can
last throughout life.
Postnatal Epigenetic Programming
(PEP)
Prenatal Epigenetic Programming
(PREP)
METHYLATION IS INHIBITED BY OXIDATIVE STRESS
Oxidative
Stress
HCY
(-)
Adenosine
A reversible reaction!
SAH
MethylTHF
Methionine
Synthase
Vitamin B12
(Cobalamin)
THF
SAM
MET
ATP
(-)
~1,000
Methylation
(+)
Reactions
via
209 MTases
PP + Pi
Global methylation =
DIETARY PROTEIN
( SAM
)
SAH
Methylation of DNA and histones is fundamental to
epigenetic regulation of gene expression during development
Regulation of gene expression during development
Transcription Factor Regulation:
Growth Factors
Neurotransmitters
Hormones
Start site for
mRNA synthesis
CpG TF CpG
TF binding region
DNA
Gene sequence
RNA
polymerase
SAM
Protein
(e.g. enzyme)
Me
MBDP
Me
HMT
Histone
proteins
(e.g. MeCP2)
DNA
Translation
X
Epigenetic Regulation:
DNMT
mRNA
Transcription
Me
Me
CpG
CpG
TF binding region
SAM
Me
Me
X No mRNA
DNA + Histone = Heterochromatin
Genes are silenced and transcription is blocked
Epigenetic changes in gene expression are the
Primary mechanism underlying development
Epigenetic marks change in response to the environment and
contribute to adaptive capabilities gained through evolution
Essentially:
Epigenetics = A memory system
• linked to gene expression
• responsive to redox status
• driver of development
• active in all cell types
• active at all ages
• capable of transgenerational effects
Agents which interrupt antioxidant
and/or methylation pathways will
interfere with the many roles of
epigenetic regulation.
PrEP
PEP
Prenatal
Epigenetic
Programming
Postnatal
Epigenetic
Programming
Maternal Metabolism
Breast Milk (Formula)
Maternal Nutrition
Toxic Exposures
Maternal
Toxic Exposures
Physical Experiences
Placental Function
Emotional Experiences
Genetic Factors
BIRTH
GI tract absorption of cysteine is critical for
postnatal epigenetic programming (PEP)
GI TRACT
BLOOD
BRAIN
Blood
Brain
Barrier
GI
Epithelial
Cells
EAAT3
Cysteine
GSH
Cysteine
Astrocytes
Cystine
GSH
GSH
Cysteine
Cystine
Methionine
Cysteine
Cystine EAAT3
GSH
Dietary
Protein
Cysteine
Neurons
LIVER
Cysteine
Cystine
Odds of autism decrease with
increasing duration of breastfeeding
Data from Waly et al. (In Prep)
Folate and B12 Levels are Markedly Lower
Data from Waly et al. (In Prep)
Thus malnutrition-based autism in Oman shares
a similar metabolic profile to autism in the U.S.
i.e. Oxidative stress and impaired methylation
are fundamental features of autism
Data from Waly et al. (In Prep)
Growth factors stimulate cysteine uptake, providing
a powerful mechanism to regulate redox and methylation
Neurotrophic
Growth Factors
Cysteine
Cysteinylglycine
Glial Cells
(Astrocytes)
GSH
EAAT3
PI3-kinase
(+)
GSSG
GSH
γ-Glutamylcysteine
EAAT3
Cysteine
Cystathionine
Adenosine
SAH
HCY
MethylTHF
Methionine
Synthase
>150
Methylation
Reactons
THF
SAM
MET
ATP
PP+Pi
(-)
FOOD-DERIVED OPIATE PEPTIDES
Beta-Casein
Human breast milk
β-Casein ( 40 % of milk protein)
59
66 -67
Wheat, barley, rye
-L- V- Y- P- F- P- G- P- I -X
Gliadins
Human β-Casomorphin -7
A1
(His at 67)
Tyr-Pro-Phe-Val-Glu-Pro-Ile
Bovine β-Casomorphin -7
(0.4 g/L milk)
Tyr-Pro-Phe-Pro-Gly-Pro-Ile
A2
(Pro at 67)
α- gliadin-7
Tyr-Pro-Gln-Pro-Gln-Pro-Phe
A1 casein contains HIS at position 67 and is readily hydrolyzed to BCM7
A2 casein contains PRO at position 67 and is resistant to hydrolysis
IC50 values
62 nM
14 nM
16 nM
4 nM
7.022 nm
Bovine casomorphin 7 decreases cysteine, glutathione,
and methionine levels in neuronal cells.
Homocysteine and cystathionine levels are increased.
This is consistent with:
1. Decreased cysteine uptake
2. Decreased GSH synthesis
3. Lower activity of methionine
synthase
4. Increased transsulfuration
Opiate peptides can inhibit cysteine uptake and promote oxidative stress
GI Tract
GI
Epithelial
Cell
Blood
Blood
Brain
Barrier
Brain
Gluten/Casein
Astrocytes
↓GSH
↓Cystine
Cysteine
Cysteine
↓Cysteine
(- )
↓Cystine
Gluten/Casein
Opiate Peptides
↓GSH
↓Cysteine Opiate
Peptides
↓Cystine
(-)
↓Cysteine
↓GSH
Oxidative Stress
↓ Methylation
Wheat/Milk
Neurons
Liver
↓Cysteine
↓Cystine
Effects of morphine and bovine and human casomorphin-7 peptides
on redox/methylation gene expression in human neuronal cells
Breast Milk Constituents Can Exert Epigenetic Effects
BREAST MILK
Casein
Growth
Factors
GI
Peptidases
DHA
Beta Casomorphin
Modulation of GI cysteine uptake
Systemic availability of cysteine
Δ Cellular Redox Status
Δ Methylation Status
Δ Gene Expression
Brain
GI Tract
Immune
System
Terminal Ileum is critical site for absorption of cysteine
and selenocysteine via the EAAT3 transporter
Terminal
Ileum
GI tract
Lumen
Body
Epigenetic Regulation
Food-derived
amino acids
Redox Status
Extracellular
Redox
Status
GSH
CYS
GSSG
GSH
Se-Proteins
CYS
Se-CYS
CYS
Se-CYS
EAAT3
GI Epithelial Cell
Terminal ileum is also critical for vitamin B12 and folic acid absorption
Terminal
Ileum
GI tract
Lumen
Body
Epigenetic Regulation
MRP1
B12/Transcobalamin II
Folic acid
Redox Status
Food-derived
B12+ Folate
GS-B12 (?)
B12/Intrinsic Factor
Folic acid
Folic acid
RFC-1
RFC-1
GI Epithelial Cell
Antioxidant Availability
(i.e. cysteine and GSH)
Metabolic Activity
(Antioxidant demand)
Homeostatic Equilibrium
↑ Metabolic Activity
(Higher antioxidant demand)
↓Antioxidant Availability
(Low cysteine and GSH)
Adaptive
Oxidative Stress → Epigenetic
Changes
Autism-associated changes in gene expression in terminal ileum
Data from Drs. Steve Walker and Arthur Krigsman
Terminal Ileum
CBS
GSS
TNFA
GSR
DRD4
GCLC
CTH
GCLM
EAAT3
MTHFR
NRF2
fold change
2.37
1.52
1.63
1.72
2.02
1.58
2.63
1.7
2.19
1.55
3.12
direction
Down
Down
Up
Down
Down
Down
Down
Down
Down
Down
Down
A preliminary qRT-PCR evaluation of redox/methylation-related
gene expression In blood-derived RNA of autistic vs. control subjects
Figure:2
Cysteine
EAAT3
Excitatory
amino acid
Transporter
GSH
Glutathione
Synthetase
(GSS)
GSSG
Glutathione
Reductase
(GSR)
γ-Glutamylcysteine
Cysteine
Gammaglutamylcysteine
synthetase
(GCLC/GCLM)
Cystine
Cystathionine
gamma-lyase
(CGL/CTH)
Cystathionine
Cystathionine
beta- synthase
(CBS)
Adenosine
Homocystine
Methionine
Synthase
(MS)
MeCbl
Methionine
Synthase
SAM
GSH
Methionine
Adenosyl
Transferase 2A
(MAT 2A)
OHCbl
GSCbl
SAH
HCY
MethylTHF
Methylated DNA
THF
DNA
SAM
MET
ATP PP+P
Methyl Transferases
Ex. DNA (cytosine-5-)methyltransferase (DNMT3A)
Ex. Catechol-Omethyltransferase (COMT)
The brain extracellular environment (CSF) has
more than 10-fold less cysteine than plasma!!
Blood-Brain
Barrier
BRAIN
Neurons
Astrocytes
[GSH] = 0.21mM
[GSH] = 0.91mM
[CYS]
BLOOD
[GSH] = 3.5 μM
[CYS] =22 μM
[CYS]
[CysGly]
[GSH]
CSF
[GSH] = 0.3 μM
12-fold lower
[CYS] = 2.2 μM
10-fold lower
Data from: Castagna et al. Neurology, 45:1678-83 (1995) and Sun et al. J Biol Chem, 281:17420-31 (2006).
Redox and Methylation Pathways in Neurons
Neurotrophic
Growth
Factors
Cysteine
Cysteinylglycine
GSH
Astrocytes
GSH
Cystine
EAAT3
PI3-kinase ( + )
GSSG
EAAT3
GSH
γ-Glutamylcysteine
Cysteine
PARTIALLY BLOCKED IN
NEURONAL CELLS
Cystathionine
Adenosine
D4SAH
Adenosine
D4HCY
SAH
HCY
MethylTHF
Phospholipid
Methylation
MethylTHF
Methionine
Synthase
THF
D4SAM
PP+Pi
ATP
Dopamine
>1,000
Methylation
Reactions
THF
D4MET
SAM
MET
ATP
(- )
PP+Pi
NEURON
MS Cob mRNA (arbitrary units)
Methionine synthase mRNA in cortex progressively
decreases with increasing age in normal subjects
600
28 weeks of
fetal development
500
400
T1/2fast = 3.4 years
T1/2slow = 29.4 years
R2 = .91
300
400-fold
200
100
0
0
10 20 30 40 50 60 70 80 90 100
Age (years)
The level of methionine synthase mRNA is reduced by 50% in
postmortem brain of autistic subjects vs. age-matched controls
Redox signaling plays a central role in the immune response
Dendritic cells release GSH,
similar to astocytes in brain
Antigen
Regulatory T-cells compete
with Teff for cysteine
and restrict the immune response
Effector T-cells take up cysteine,
similar to neurons in brain
Antibody
production
EAAT3 provides
Cysteine uptake
EAAT3 is expressed in T cell lymphocytes
and highest expression is in Treg cells
Autoimmune-prone SJL/J mice have lower EAAT3
expression in mixed T cell lymphocytes
EAAT3-mediated cysteine/selenocysteine
uptake in the terminal ileum
EAAT3
Autoimmune-prone SJL/J mice have lower levels of GSH
in brain, unaffected by postnatal thimerosal treatment
Autoimmune-prone SJL/J mice have lower methionine synthase
activity in brain, unaffected by postnatal thimerosal treatment
A1 Beta Casein Consumption is correlated
with increased risk of juvenile-onset diabetes
r = 0.92
Using A1 cow milk formula with
hydrolyzed casein lowered
autoimmunity and incidence
of type I diabetes by ~ 50%
GI uptake of cysteine is critical throughout life, but
especially during postnatal epigenetic programming (PEP)
Gluten
Casein
Autism
Opiate
Peptides
↓Cysteine Uptake
Autoimmunity
(Type 1 diabetes)
GI Inflammation
(celiac disease)
↓ Antioxidant Capacity
ACKNOWLEDGEMENTS
Brain Samples:
Autism Tissue Program
Harvard Brain Tissue Resource Center
Tissue Resource Center (Australia)
Stanley Medical Research Foundation
and donor families.
Collaborators:
Sultan Qaboos University
Mostafa Waly and Yahya Al-Farsi
Grant Support:
Autism Research Institute
SafeMinds
National Autism Association
Autism Speaks